Phospholipase A2s (PLA2s) are an expanding super family of esterases that cleave the acyl ester bond at the sn-2 position of membrane phospholipids to produce a free fatty acid and lysophospholipid (Farooqui et al., 2000, Neuroscientist 6:169-180). Because a large proportion of cellular arachidonic acid is found esterified at the sn-2 position of membrane phospholipids, arachidonic acid and lysophospholipid are the major products of the PLA2-catalyzed reaction. Under normal conditions, some arachidonic acid is converted to inflammatory mediators, prostaglandins, leukotrienes, and thromboxanes, whereas a majority of arachidonic acid is reincorporated into brain phospholipids (Rapoport, 1999, Neurochem. Res. 24:1403-1415; Leslie, 2004, Biochem. Cell. Biol. 82:1-17). Arachidonic acid not only acts via conversion to inflammatory metabolites, but can also directly modulate neuronal function by various mechanisms, such as altering membrane fluidity and polarization state, activating protein kinase C, and regulating gene transcription (Katsuki and Okuda, 1995, Prog. Neurobiol. 46:607-636; Farooqui et al., 1997 Arachidonic acid, neurotrauma, and neurodegenerative disease, in Handbook of Essential Fatty Acid Biology (Yehuda and Mostofsky, eds.) pp 277-295, Humana Press, Totowa, N.J.). Another product of PLA2 catalyzed reactions, 1-alkyl-2-lysophospholipid, is the immediate precursor of platelet-activating factor (PAF), another potent inflammatory mediator (Farooqui and Horrocks, 2004, Plasmalogens, platelet activating factor, and other lipids, in Bioactive Lipids (Nicolaou and Kokotos, eds.) pp 107-134, Oily Press, Bridgwater, U.K.).
Increased PLA2 activity and excessive production of proinflammatory mediators, eicosanoids, and platelet activating factor, may potentially lead to disease states and neuronal injury. PLA2-generated mediators play a central role not only in acute inflammatory responses in brain but also in oxidative stress associated with progressive degenerative neurological disorders such as Alzheimer's disease (AD), Parkinson's disease (PD), and multiple sclerosis (MS) (Kalyvas and David, 2004, Neuron 41:323-335; Phillis and O'Regan, 2004, Brain Res. Rev. 44:13-47; Sun et al., 2004, J. Lipid Res. 45:205-213). PLA2 contributes to the pathogenesis of the above disorders by attacking neural membrane phospholipids and releasing proinflammatory lipid mediators such as prostaglandins, leukotrienes, and thromboxanes, and PAF, and also by generating 4-hydroxynonenal (4-HNE).
Secretory phospholipase A2 (sPLA2) is synthesized intracellularly, then secreted from the cell where it acts extracellularly. PLA2 binds to two types of cell surface receptors, namely the N type in neurons, and the M type in skeletal muscles, (Hanasaki and Arita, 2002, prostaglandins Other Lipid Mediat. 68-69:71-82). Brain sPLA2 is present in all regions of mammalian brain with the highest activities of sPLA2 are found in medulla oblongata, pons, and hippocampus, moderate activities in the hypothalamus, thalamus, and cerebral cortex, and low activities in the cerebellum and olfactory bulb (Thwin et al., 2003, Exp. Brain Res, 150:427-433).
Glutamate and its analogs stimulate sPLA2 activity in a dose- and time-dependent manner (Kim et al., 1995, Biochem. J. 310:83-90; Xu et al., 2003, Free Radical Biol. Med. 34:1531-1543). The neurotoxicity of glutamate is synergistically increased with the addition of sPLA2 to cortical cultures, suggesting glutamatergic synaptic activity may be modulated by sPLA2 and its receptors on the neuronal surface (DeCoster et al., 2002, J. Neurosci. Res. 67:634-645; Kolko et al., 2002, NeuroReport 13:1963-1966). In PC12 cells, sPLA2 induces neurite outgrowth. Mutants with reduced sPLA2 activity exhibit a comparable reduction in neurite-inducing activity (Nakashima et al., 2003, Biochem. J. 376:655-666), indicating that sPLA2 performs a neurotrophin-like role in the central nervous system.
Neurons are more susceptible to free radical-mediated neuroinflammation and oxidative stress than glial cells (Adibhatla et al., 2003, J. Neurosci. Res. 73:308-315; Ajmone-Cat et al., 2003, J. Neurochem. 87:1193-1203). In fact, activated glial cells, including astroglia and microglia, sustain inflammatory processes initiated by arachidonic acid-generated metabolites. This suggests that signals modulating the induction, expression, and stimulation of PLA2 isoforms may play an important role in neurodegenerative diseases associated with Neuroinflammation and oxidative stress (Farooqui and Horrocks, 1994, Int. Rev. Neurobio, 36:267-323; Farooqui et al., 2003, Stimulation of lipases and phospholipases in Alzheimer disease, in Nutrition and Biochemistry of Phospholipids (Szuhaj and van Nieuwenhuyzen, eds.) pp 14-29 AOCS Press, Champaign, Ill.; Farooqui and Horrocks, 2004, Plasmalogens, platelet activating factor, and other lipids, in Bioactive Lipids (Nicolaou and Kokotos, eds.) pp 107-134, Oily Press, Bridgwater, U.K.). For the successful treatment of inflammatory and oxidative stress in neurological disorders, timely delivery of a well-tolerated, chronically active, and specific inhibitor of PLA2 that can bypass or cross the blood-brain barrier without harm is required. Some nonspecific PLA2 inhibitors have been used for the treatment of neurological disorders such as ischemia, spinal cord injury, and AD (Sano et al., 1997, New Eng. J. med. 336:1216-1222), but no compound with real clinical potential has emerged.
The neuron survival-promoting peptide Y-P30 was originally identified in the secretions of neural cells (neuroblastoma and retinoblastoma) subjected to oxidative stress (Cunningham, et al. 1998, J. Neurosci. 18:7047-7060). Partially purified fractions of conditioned culture medium were screened in vitro until the active Y-P30 peptide was identified—the synthetic version of this peptide was then tested in vitro and in vivo and found to support neural cells which were degenerating for a variety of reasons, including oxidative stress and central nervous system trauma (Cunningham, et al. 1998, J. Neurosci. 18:7047-7060; Cunningham et al., 2000, Exp. Neurol. 163:457-468). This peptide was later confirmed to be part of an endogenous human polypeptide (−12 kiloDaltons) named DSEP after identification of the human cDNA encoding DSEP and the locus of the DSEP gene in human chromosomal region 12q (Cunningham, et al. 2002, Exp. Neurol. 177:32-39). In that study, it was found that overexpression of the full length polypeptide in neural cells made them resistant to several forms of oxidative stress including that resulting from immune cell attack. CHEC-9 and CHEC-7 are anti-inflammatory and neuron survival-promoting peptides that inhibit enzymes that initiate a cascade of changes during the early stages of inflammation.
The stimulation of sPLA2 and subsequent biochemical cascade are important events associated with acute neural trauma as well as chronic neurological degenerative diseases (Farooqui et al., 2006, Pharmacological Rev. 58:591-620). Similarly, atherosclerosis has been proposed as both a disorder of inflammation and lipid metabolism (Jaross et al., 1999, Atherosclerosis 144 (Supplement 1):119-120). The sPLA2s are a subclass of phospholipase A2 enzymes that cleave the A2 fatty acid ester of phospholipids (Burke et al, 2009, J. Lipid Res. 50:S237-242). Several sPLA2s have been identified and the contribution of specific enzyme isoforms (principally groups II, V and X) and play a role in the formation of atherosclerotic lesions (Rosenson, 2009, Cardiovascular Drugs and Therapy 23:93-101). sPLA2 inhibitors are attractive therapeutic targets. However, existing and available sPLA2 inhibitors lack sufficient specificity, affecting not just other PLA2 isoforms, but other enzymes such as cyclooxygenase and acyltransferase (Cummings et al., 2000, J. Pharmacal. Exp. Ther. 294:793-799; Fuentes et al., 2003, J. Biol. Chem. 278:44683-44690).
There is a long standing need in the art for specific and potent sPLA2 inhibitors that are well-tolerated clinically for use in methods of treating a variety of non-degenerative neurological diseases or disorders and cardiovascular diseases. The present invention fills this need.